Hydration of protein–RNA recognition sites

We investigate the role of water molecules in 89 protein–RNA complexes taken from the Protein Data Bank. Those with tRNA and single-stranded RNA are less hydrated than with duplex or ribosomal proteins. Protein–RNA interfaces are hydrated less than protein–DNA interfaces, but more than protein–protein interfaces. Majority of the waters at protein–RNA interfaces makes multiple H-bonds; however, a fraction do not make any. Those making H-bonds have preferences for the polar groups of RNA than its partner protein. The spatial distribution of waters makes interfaces with ribosomal proteins and single-stranded RNA relatively ‘dry’ than interfaces with tRNA and duplex RNA. In contrast to protein–DNA interfaces, mainly due to the presence of the 2′OH, the ribose in protein–RNA interfaces is hydrated more than the phosphate or the bases. The minor groove in protein–RNA interfaces is hydrated more than the major groove, while in protein–DNA interfaces it is reverse. The strands make the highest number of water-mediated H-bonds per unit interface area followed by the helices and the non-regular structures. The preserved waters at protein–RNA interfaces make higher number of H-bonds than the other waters. Preserved waters contribute toward the affinity in protein–RNA recognition and should be carefully treated while engineering protein–RNA interfaces.     

Identification of β-aggregate sites in protein chains

The problem of amyloidoses is pressing and have recently attracted special attention throughout the world because of epidemics of prion diseases such as mad cow disease and human Creutzfeldt-Jacob disease. These diseases result from the conversion of a native protein or peptide into a highly stable pathological form. Molecules having a pathological conformation aggregate to form amyloid fibrils, capable of unlimited growth. It is important to study the molecular mechanisms of prion diseases and to identify the protein regions responsible for their development. The review considers theoretical and experimental works focusing on the formation of amyloid fibrils.     

Homology model and docking studies on porcine β<Subscript>2</Subscript> adrenoceptor: description of two binding sites

The affinity of the classical β₂ adrenoceptor-selective inverse agonist ICI118,551 is notoriously lower for porcine β₂ adrenoceptors (p₂βAR) than for human β₂ adrenoceptors (hβ₂AR) but molecular mechanisms for this difference are still unclear. Homology 3-D models of pβ₂AR can be useful in predicting similarities and differences, which might in turn increase the comparative understanding of ligand interactions with the hβ₂AR. In this work, the pβ₂AR amino acid sequence was used to carry out homology modeling. The selected pβ₂AR 3-D structure was structurally and energetically optimized and used as a model for further theoretical study. The homology model of pβ₂AR has a 3-D structure very similar to the crystal structures of recently studied hβ₂AR. This was also corroborated by sequence identity, RMSD, Ramachandran map, TM-score and docking results. Upon performing molecular docking simulations with the AutoDock4.0.1 program on pβ₂AR, it was found that a set of well-known β₂AR ligands reach two distinct binding sites on pβ₂AR. Whereas one of these sites is similar to that reported on the hβ₂AR crystal structure, the other can explain some important experimental observations. Additionally, the theoretical affinity estimated for ICI118,551 closely agrees with affinities estimated from experimental in vitro data. The experimental differences between the human/porcine β₂ARs in relation to ligand affinity can in part be elucidated by observations in this molecular modeling study.     

Spectrin-phospholipid interactions. Existence of multiple kinds of binding sites?

The object of this paper is to review briefly the studies on the interactions of erythroid and non-erythroid spectrins with lipids in model and natural membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Another important issue is the occurrence of spectrins in membrane rafts, how they are attached to the raft and what is their function in rafts.     

Heterodimers of wild‐type and subunit interface mutant enzymes of glutathione S‐transferase A1–1: Interactive or independent active sites?

Heterodimers of rat glutathione S-transferase A1-1 were formed using one wild-type subunit and one subunit with a mutation at the interface to evaluate whether the subunits are interactive or independent. Within the subunit interface, we are considering two regions of interactions: one region consists of a "hydrophobic ball and socket" with Phe 52 from one subunit as the ball and Phe 136 from the second subunit as one of the socket residues. The second region of interaction consists of Arg 69 and Glu 97 from both subunits. The heterodimers were formed after incubation in 1,6-hexanediol. Because one subunit in each pair had a His-tag, the heterodimers were purified using a nickel-nitrilotriacetic acid column. The specific activities of the heterodimer were compared with those of the two homodimers to determine whether the less active, mutant subunit communicates with the other subunit. Two of the heterodimers, wild type/R69E-His and wild type/E97Q-His, displayed specific activities much lower than that expected for independent active sites; in these cases, there are new close repulsive interactions and the low activity of one subunit is communicated to the neighboring subunit. In contrast, the other two heterodimers, wild type/R69Q-His and F136A/wild type-His, exhibited specific activities similar to those expected for independent active sites; in these heterodimers, the closest interaction is not repulsive or occurs over a much longer distance and the subunits act independently. We conclude that whether the subunits interact or are independent depends on the nature of the interactions at the subunit interface.     

Evidence of multiple causal sites affecting weight in the <Emphasis Type="Italic">IGF2-INS-TH</Emphasis> region of human chromosome 11

The subtelomeric region of 11p harbours three closely linked genes, TH, INS and IGF2, that have been associated with obesity, size at birth, type I diabetes, polycystic ovary syndrome, overgrowth in Beckwith-Wiedemann syndrome and possibly hypertension. We have previously shown that the IGF2 ApaI single nucleotide polymorphism (SNP) associates with weight and body mass index in middle-aged Caucasian males but that there is no such association with the INS -23/ HphI site that marks INS 5' variable number of tandem repeats (VNTR) class I vs class III VNTR alleles. We report here the examination of three SNP markers in IGF2: 6815 A/T in the P1 promoter, AluI in exon 3 and ApaI in the 3' untranslated region (UTR), INS 5'VNTR class I alleles and the TH01 tetranucleotide microsatellite in a population sample. The analysis has taken into account the possibility that typing failure and the number of parameters required to model multiallelic loci could create spurious significance. We have exercised Hardy-Weinberg equilibrium tests, dichotomised multiallelic series to impose parsimony, and examined the data with failures modelled or excluded. Regression analysis infers that three markers, IGF2 ApaI, TH01 and subclasses of INS VNTR class I independently predict derived weight indices (combined P<10(-8) and accounting up to 2% of population weight variance), with no evidence of interaction. This establishes that there must be multiple causal sites impacting on weight in this genomic region.     

Internalization of β-adrenergic receptor binding sites: Involvements of lysosomal enzymes

Lysosomal enzymes were detected in a highly purified preparation of frog erythrocytes. Pretreatment of intact erythrocytes with lysosomotropic drugs reduced the number of soluble β-receptors in isoproterenol-treated cells, whereas the level of membrane-bound receptors in these cells was unaffected. Subcellular fractionation by Percoll gradient centrifugation revealed that one species of lysosomes (density: 1.15 g/ml), contained a fraction of membrane-bound β-adrenergic receptors. This fraction of membrane-bound receptors was markedly increased when desensitized cells were pretreated with chloroquine. Thus the internalized receptors appear to be delivered to lysosomes and released from the endocytic vesicles by the lysosomal enzymes.     

Prior knowledge or freedom of interpretation? A critical look at a recently published classification of “novel” Zn binding sites

In a recently published article (Yao, Flight, Rouchka, and Moseley, Proteins 2015;83:1470–1487) the authors proposed novel Zn coordination patterns in protein structures, apparently discovered using an unprejudiced approach to the information collected in the Protein data Bank (PDB), which they advocated as superior to the prior‐knowledge‐informed paradigm. In our assessment of those propositions we demonstrate here that most, if not all, of the “new” coordination geometries are fictitious, as they are based on incorrectly interpreted protein crystal structures, which in themselves are often not error‐free. The flaws of interpretation include partial or wrong Zn sites, missed or wrong ligands, ignored crystal symmetry and ligands, etc. In conclusion, we warn against using this and similar meta‐analyses that ignore chemical and crystallographic knowledge, and emphasize the importance of safeguarding structural databases against bad apples. Proteins 2016; 84:770–776. © 2016 Wiley Periodicals, Inc.     

X-ray absorption and diffraction studies of the metal binding sites in amyloid <Emphasis Type="Italic">β</Emphasis>-peptide

A major source of neurodegeneration observed in Alzheimer’s disease is believed to be caused by the toxicity from reactive oxygen species produced in the brain mediated by the Aβ protein and mainly copper species. An atomic model of an amyloid β-peptide (Aβ) Cu²⁺ complex or at least the structure of the metal binding site is of great interest. Accurate information about the Cu-binding site of Aβ protein can facilitate simulation of redox chemistry using high level quantum mechanics. Complementary X-ray diffraction and X-ray absorption techniques can be employed to obtain such accurate information. This review provides a blend of X-ray diffraction results on amyloid structures and selected works on Aβ Cu²⁺ binding based on spectroscopic measurements with emphasis on the X-ray absorption technique. Australian Society for Biophysics Special Issue: Metals and Membranes in Neuroscience.     

Micro-mechanical properties of different sites on woodpecker’s skull

The uneven distributed microstructure featured with plate-like spongy bone in woodpecker’s skull has been found to further help reduce the impact during woodpecker’s pecking behavior. Therefore, this work was to investigate the micro-mechanical properties and composition on different sites of Great Spotted woodpecker’s (GSW) skull. Different sites were selected on forehead, tempus and occiput, which were also compared with those of Eurasian Hoopoe (EH) and Lark birds (LB). Micro structural parameters assessed from micro computed tomography (μCT) occurred significantly difference between GSW, EH and LB. The micro finite element (micro-FE) models were developed and the simulation was performed as a compression process. The maximal stresses of GSW’s micro-FE models were all lower than those of EH and LB respectively and few concentrated stresses were noticed on GSW’s trabecular bone. Fourier transform infrared mapping suggesting a greater organic content in the occiput of GSW’s cranial bone compared with others. The nano-hardness of the GSW’s occiput was decreasing from forehead to occiput. The mechanical properties, site-dependent hardness distribution and special material composition of GSW’s skull bone are newly found in this study. These factors may lead to a new design of bulk material mimicking these characteristics.     

Autoradiographic localization of α-bungarotoxin-binding sites in the carotid body of the rat

Summary Radioiodinated -bungarotoxin (-Bgt) was used to localize -Bgt-acetylcholine receptors in the carotid body of the rat. The gamma spectrometer analyses indicated a high uptake of [¹²⁵I] -Bgt in carotid bodies incubated in vitro (1.51 fmole per organ). Incorporation of the isotope was effectively blocked by pretreatment of carotid bodies with d-tubocurarine and unlabeled -Bgt, but not by atropine. Light microscopic autoradiography showed a heavy labeling of some parenchymal cells. Electron-microscopic autoradiography revealed that labeling was localized along the interface between parenchymal cells, especially where their cytoplasmic processes engage in complex interdigitations. The silver grain counts on electron-microscopic autoradiographs suggest that labelings are preferentially associated with the plasma membrane of certain Type I cells. It is suggested that these Type I cells in the rat's carotid body probably are provided with nicotinic acetylcholine receptors on their plasma membranes.This research was partly supported by a grant from the Edward G. Schleider Foundation of New Orleans.The authors extend appreciation to Drs. Akira Arimura, Dept. of Medicine, for supplying a part of the radioisotope and to James Fisher, Dept. of Pharmacology, Tulane Medical School, for use of the gamma spectrometer. Appreciation also is extended to Mrs. Lia Pedroza for technical assistance     

Membrane traffic: do cones mark sites of fission?

Membrane fission occurs in eukaryotic cells whenever a vesicle is produced or a larger subcellular compartment is divided into smaller discrete units. Recent evidence suggests this fission event is promoted by enzymes that generate phosphatidic acid and thereby cause a distortion of the lipid bilayer.     

Multiplicity of carbohydrate-binding sites in <Emphasis Type="Italic">β</Emphasis>-prism fold lectins: occurrence and possible evolutionary implications

The β-prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit/domain. Until recently, β-prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit/domain. However, the recently determined structure of the β-prism fold I lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for two-lectin folds and which carry one or more relevant carbohydrate-binding motifs. The very recent observation of a β-prism I fold lectin, griffithsin, with three binding sites in each domain further confirmed the need for such an analysis. The analysis demonstrates substantial diversity in the number of binding sites unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of β-prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion. It would seem that the preponderance of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the β-prism II fold, is related to the role of plant lectins in defence.     

Refuge sites of voles under owl predation risk: priority of dominant individuals?

In an aviary experiment, we studied whether body size or habitatfamiliarity of field voles (Microtus agrestis) affected predationrisk by Tengmalm's owls (Aegolius funereus). In the field, wecompared the body size of field voles snap-trapped in good (covered)and poor (open) habitats in 1992 and 1994 to determine whetherthere were habitat-related differences in the body size of voles.In the aviary, large individuals occupied the good habitat significantlymore than small individuals both in the control (owl not present)and experimental treatments (owl present). Furthermore, habitat-familiarvoles inhabited the good habitat more than habitat-unfamiliarvoles did when an owl was present Our field data were consistentwith our aviary data: larger field voles were more frequentlyfound in good habitats than in poor habitats. In the aviary,Tengmalm's owl predation risk was higher for small and habitat-unfamiliarvoles. This suggests that large field voles may have priorityto sheltered habitats. Furthermore, habitat familiarity mayplay a central role in avoiding risky habitats.     

A new method for estimating the C → T transition rate in methylation sites of <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

A high-throughput method based on the minisequencing reaction followed by MALDI-TOF mass spectrometry has been developed for detecting C → T transitions in 291 methylation sites of M.Hpy99XI Helicobacter pylori J99. The study has shown an absolute (100%) accuracy of the new method.     

Identification of new Gβγ interaction sites in adenylyl cyclase 2

The role of Gβγ in adenylyl cyclase (AC) signaling is complicated due to its role as a conditional activator (AC2, AC4 and AC7) and an inhibitor (AC1, AC3 and AC8). AC2 is stimulated by Gα_s and if Gβγ is present the stimulation is synergistic. The precise mechanism of this synergistic activation is still not known. In order to further elucidate the role of Gβγ in AC2 activation by Gα_s, peptides derived from the C1 domains of AC2 were synthesized and the ability of the various peptides to regulate AC2 function was tested. Our results identify two new Gβγ-binding sites in the AC2 C1 domain, AC2 C1a 339-360 and AC2 C1b 578-602 that are involved with stimulation of AC2 by Gβγ. These two regions are different from the previously described QEHA motif in the C2 domain of AC2. Further, the recently discovered PFAHL motif was confirmed to bind and to be involved with stimulation of AC2 by Gβγ. These functional studies indicate that multiple regions of AC2 are involved in the interaction with Gβγ.     

Muslim community organizations – sites of active citizenship or self-segregation?

Ethno-religious community organizations in Western countries have often been described as being disconnected from mainstream society, and Muslim community groups have been a special focus of such critique. This article offers a counter-narrative to these widespread allegations. It draws on a synthesis of emerging research on the citizenship-enhancing effects of mosque involvement and on an explorative study involving thirty in-depth interviews with civically active Muslims in Australia and Germany. The article examines the potential of Muslim community organizations to mobilize their member into performing their citizenship through civic and political participation. It offers empirical evidence that many Muslim community organizations, rather than promoting social segregation, act as accessible entry point for Muslims’ civic participation, facilitate cross-community engagement and provide gateways to political involvement. These civic potentials of Muslim community organization have remained underestimated in the public and political discourse on cohesive societies and healthy democracies.